THE ANATOMICAL RECORD 291:263–282 (2008) Volumetric Neuroimaging of the Atlantic White-Sided Dolphin (Lagenorhynchus acutus) Brain From In Situ Magnetic Resonance Images ERIC W. MONTIE,1* GERALD SCHNEIDER,2 DARLENE R. KETTEN,1 3 4 1 LORI MARINO, KATIE E. TOUHEY, AND MARK E. HAHN * 1Department of Biology, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 2Department of Brain and Cognitive Sciences, MIT, Cambridge, Massachusetts 3Neuroscience and Behavioral Biology Program, Emory University, Atlanta, Georgia 4Cape Cod Stranding Network, Buzzards Bay, Massachusetts ABSTRACT The structure and development of the brain are extremely difficult to study in free-ranging marine mammals. Here, we report measurements of total white matter (WM), total gray matter (GM), cerebellum (WM and GM), hippocampus, and corpus callosum made from magnetic resonance (MR) images of fresh, postmortem brains of the Atlantic white-sided dol- phin (Lagenorhynchus acutus) imaged in situ (i.e., the brain intact within the skull, with the head still attached to the body). WM:GM volume ratios of the entire brain increased from fetus to adult, illustrating the increase in myelination during ontogeny. The cerebellum (WM and GM combined) of subadult and adult dolphins ranged from 13.8 to 15.0% of total brain size, much larger than that of primates. The corpus callosum mid-sagittal area to brain mass ratios (CCA/BM) ranged from 0.088 to 0.137, smaller than in most mammals. Dolphin hippocampal volumes were smaller than those of carnivores, ungulates, and humans, consistent with previous qual- itative results assessed from histological studies of the bottlenose dolphin brain. These quantitative measurements of white matter, gray matter, cor- pus callosum, and hippocampus are the first to be determined from MR images for any cetacean species. We establish here an approach for accu- rately determining the size of brain structures from in situ MR images of stranded, dead dolphins. This approach can be used not only for compara- tive and developmental studies of marine mammal brains but also for investigation of the potential impacts of natural and anthropogenic chemi- cals on neurodevelopment and neuroanatomy in exposed marine mammal populations. Anat Rec, 291:263–282, 2008. Ó 2008 Wiley-Liss, Inc. Key words: cetacean; dolphin; MRI; white matter; cerebellum; corpus callosum; hippocampus; brain Grant sponsor: Environmental Protection Agency; Grant E-mail: [email protected] or Eric W. Montie, College of Marine number: U-91616101-2; Grant sponsor: the National Woman’s Science, University of South Florida, 140 Seventh Avenue, Farm and Garden Association; Grant sponsor: the Quebec Labrador South, St. Petersburg, FL 33701-5016. Fax: 727-553-1193. Fund/Atlantic Center for the Environment; Grant sponsor: Woods E-mail: [email protected] Hole Oceanographic Institution; Grant sponsor: the Sawyer Received 17 October 2007; Accepted 3 December 2007 Endowment; Grant sponsor: Walter A. and Hope Noyes Smith. DOI 10.1002/ar.20654 Dr. Montie’s present address is College of Marine Science, Published online in Wiley InterScience (www.interscience.wiley. University of South Florida, 140 Seventh Avenue, South, St. com). Petersburg, FL 33701-5016. *Correspondence to: Mark E. Hahn, Department of Biology, WHOI, MS#32, Woods Hole, MA 02543. Fax: 508-457-2134. Ó 2008 WILEY-LISS, INC. 264 MONTIE ET AL. Odontocetes (toothed whales, dolphins, and porpoises) validate our techniques by determining if MRI coupled have undergone unique anatomical adaptations to an with advanced software image processing and segmenta- aquatic environment. One significant modification is in tion could accurately determine volumes, (b) determine brain size. Several odontocete species have encephaliza- the white matter and gray matter volumes of the total tion quotients (a measure of relative brain size) that are brain and cerebellum along an ontogenetic series using second only to modern humans (Ridgway and Brownson, MR images, and (c) from MR images, determine the 1984; Marino, 1998). Several studies of odontocete mid-sagittal area of the corpus callosum and the vol- neuroanatomy have been completed, as reviewed by umes of the left and right hippocampal formation. Morgane et al. (1986) and Ridgway (1990). However, few studies have focused on quantitative measurements of MATERIALS AND METHODS odontocete brain structures (Tarpley and Ridgway, 1994; Marino et al., 2000). Fewer studies have focused on Specimens odontocete prenatal neuroanatomy or provided quantita- The Atlantic white-sided dolphin specimens used in tive data on prenatal brain structures (Marino et al., this study stranded live on the beaches of Cape 2001a). Cod, Massachusetts, between 2004 and 2005 (Table 1). Magnetic resonance imaging (MRI) has recently been Stranded animals were usually first reported by the used to study the neuroanatomy of the Atlantic white- public and then responded to by the Cape Cod Stranding sided dolphin (Lagenorhynchus acutus), the beluga Network (CCSN) in Buzzards Bay, MA. The specimens whale (Delphinapterus leucas), the fetal common dolphin were either found freshly dead or were humanely eutha- (Delphinus delphis), the bottlenose dolphin (Tursiops nized by stranding response personnel or by local veteri- truncatus), the harbor porpoise (Phocoena phocoena), the narians because of poor health. Less than 24 hr had dwarf sperm whale (Kogia simus), the spinner dolphin passed since the time of death in all cases. Euthanasia (Stenella longirostris orientalis), and the killer whale of these animals was approved by the National Marine (Orcinus orca; Marino et al., 2001a–c, 2003a,b, 2004a,b; Fisheries Service (NMFS) Marine Mammal Health and Montie et al., 2007). MRI offers a nondestructive method Stranding Response Program (MMHSRP). The use of of acquiring a permanent archive of external and inter- these specimens for MRI scanning and brain studies nal brain structure data. MRI coupled with advanced was approved by the Institution Animal Care and Use software image analysis can accurately determine re- Committee (IACUC) at the Woods Hole Oceanographic gional brain volumes, while traditional dissection and Institution (WHOI). photography may introduce error in performing quanti- Upon death or retrieval, the specimens were immedi- tative measurements. ately transported to the WHOI necropsy facility where Quantitative measurements of the size of brain struc- total body weights and morphometric measurements tures can be used not only to study comparative anat- were recorded. Specimens were then prepared for MRI. omy and development of cetacean brains but also to The headcoil of the MRI scanners had a circumference investigate emerging threats to marine mammals. These of 80 cm. Therefore, the blubber, nuchal fat, and semi- include anthropogenic chemicals such as hydroxylated spinalis muscle of specimens that had an axillary girth polychlorinated biphenyls (OH-PCBs; Sandala et al., greater than 80 cm were removed from the head region. 2004; Houde et al., 2006; McKinney et al., 2006) and poly- The pectoral and dorsal fins were removed from all car- brominated diphenyl ethers (PBDEs; De Boer et al., casses. The specimens were then washed, dried, and 1998), as well as biotoxins from harmful algal blooms placed in transport bags with ice surrounding the head. (Scholin et al., 2000). These chemicals can target the The specimens were then immediately transported to brain (Viberg et al., 2003; Kimura-Kuroda et al., 2005; the MRI facility or temporarily stored at 48C until imag- Silvagni et al., 2005). For example, domoic acid (a type ing could be completed. The time of the MRI was of biotoxin produced by some diatom Pseudo-nitzschia recorded. After imaging, the specimen was transported species and associated with harmful algal blooms) is back to WHOI and stored at 48C overnight. A complete neurotoxic and has been shown to cause bilateral hippo- necropsy was performed the next day. The brain was campal atrophy in California sea lions (Silvagni et al., removed, weighed, and archived whole in 10% neutral 2005). It is possible that biotoxins and environmental buffered formalin or at 2808C. pollutants cause subtle differences in the size of brain The specimens were classified as fetuses, neonates structures that are not detectable to the unaided eye; (126 cm to 140 cm), subadults (defined as reproductively therefore, volumetric neuroimaging would be a valuable immature, i.e., females of body length from 141 to 201 cm, approach to identify subtle abnormalities. However, and males of body length from 141 to 210 cm), or adults. there is a lack of information about normative size Total length measurements were used in this classifica- ranges and developmental patterns for cetacean brain tion, consistent with those previously determined by Ser- structures that may be sensitive to these etiologic geant et al. (1980). In addition, reproductive state (lacta- agents. tion and pregnancy indicated sexual maturity for Previously, we presented the first anatomically labeled females) and measurement of gonads (weight and macro- MRI-based atlas of the subadult and fetal brain of the scopic examination) also helped in classification of the Atlantic white-sided dolphin from in situ MR images of specimens into the appropriate age class. Teeth were fresh, postmortem brains intact, within the skull, with archived for future aging of dolphins. the head still attached to the body (Montie et al., 2007). Our goal in the present study was to establish a quanti- Magnetic